HIV-1 reverse transcriptase (RT) is a key target for antiretroviral drug development. To date, 12 RT inhibitors (RTIs) have been approved for the treatment of HIV-1 infection. These include the nucleoside/tide RT inhibitors (NRTI) that block HIV-1 replication by acting as chain-terminators of DNA synthesis, and the nonnucleoside RT inhibitors (NNRTI) that are allosteric inhibitors of HIV-1 RT DNA polymerization reactions. Although combination therapies that contain 2 or more RTI have reduced morbidity and mortality from HIV-1 infection, their long-term efficacy is limited by the selection of drug-resistant variants of HIV-1. A better understanding of the mechanisms involved is needed to prevent and manage drug resistance effectively. HIV-1 RT is a heterodimer composed of a 66kDa subunit (p66), and a p66-derived 51kDa subunit (p51). The catalytically active p66 subunit of RT consists of DNA polymerase (residues 1-315), connection (residues 316-427), and RNase H domains (residues 428-560). Most of the RTI resistance mutations identified to date map to the polymerase domain of RT. This is largely because the connection and RNase H domains have not been routinely analyzed in clinical samples. In fact, none of the genotyping assays available for patient management sequence the entire coding region of RT. However, a growing body of evidence has emerged that implicates mutations outside of the polymerase domain of RT in RTI resistance. For example, we were part of a multi-disciplinary study that identified the N348I mutation in the connection domain of RT that confers resistance to both NRTI and NNRTI. N348I is highly prevalent in RTI-experienced patients, occurs early in therapy (oftentimes before recognized polymerase domain mutations), and is associated with a greater increase in viremia than any of the recognized thymidine analog mutations that confer AZT resistance. In this application, we propose in-depth virology, biochemical and genotypic studies to determine the role of N348I and other candidate mutations in the C-terminal domains of HIV-1 RT in RTI resistance. This will be accomplished through 3 Specific Aims. In Aim 1, we will investigate the clinical relevance of mutations in the connection and RNase H domains by studying RTs in plasma samples from patients on RTI therapy. In Aim 2, we will elucidate the molecular mechanism(s) by which N348I and other clinically-relevant mutations in the C- terminal domains of HIV-1 RT confer NRTI and/or NNRTI resistance. These studies will provide novel insights into how the entire RT molecule (and not just the polymerase domain) functions to confer drug resistance. In Aim 3, we will combine structure-activity relationship studies with molecular modeling to gain structural insight into how mutations - that may be distal to the enzyme's active sites, nucleic acid binding tract or NNRTI- binding pocket - confer RTI resistance. In addition to providing new insights into the mechanisms of RTI resistance, the proposed studies could have important implications for the future design of genotype and phenotype tests for RTI resistance, and for identifying more effective RT inhibitors and inhibitor combinations. PUBLIC HEALTH RELEVANCE: The goal of this project is to determine the role on N348I and other mutations in the connection and ribonuclease H domains of HIV-1 reverse transcriptase (RT) in RT inhibitor resistance. The results from this study will provide timely information on a rapidly emerging area of research in HIV-1 drug resistance that is likely to provide new mechanistic insights and to influence the design and interpretation of drug resistance assays used in clinical practice.